title: Genetic variation in the Staphylococcus aureus 8325 strain lineage revealed by whole-genome sequencing.
abstract: Staphylococcus aureus strains of the 8325 lineage, especially 8325-4 and derivatives lacking prophage, have been used extensively for decades of research. We report herein the results of our deep sequence analysis of strain 8325-4. Assignment of sequence variants compared with the reference strain 8325 (NRS77/PS47) required correction of errors in the 8325 reference genome, and reassessment of variation previously attributed to chemical mutagenesis of the restriction-defective RN4220. Using an extensive strain pedigree analysis, we discovered that 8325-4 contains 16 single nucleotide polymorphisms (SNP) arising prior to the construction of RN4220. We identified 5 indels in 8325-4 compared with 8325. Three indels correspond to expected Φ11, 12, 13 excisions, one indel is explained by a sequence assembly artifact, and the final indel (Δ63bp) in the spa-sarS intergenic region is common to only a sub-lineage of 8325-4 strains including SH1000. This deletion was found to significantly decrease (75%) steady state sarS but not spa transcript levels in post-exponential phase. The sub-lineage 8325-4 was also found to harbor 4 additional SNPs. We also found large sequence variation between 8325, 8325-4 and RN4220 in a cluster of repetitive hypothetical proteins (SA0282 homologs) near the Ess secretion cluster. The overall 8325-4 SNP set results in 17 alterations within coding sequences. Remarkably, we discovered that all tested strains of the 8325-4 lineage lack phenol soluble modulin α3 (PSMα3), a virulence determinant implicated in neutrophil chemotaxis, biofilm architecture and surface spreading. Collectively, our results clarify and define the 8325-4 pedigree and reveal clear evidence that mutations existing throughout all branches of this lineage, including the widely used RN6390 and SH1000 strains, could conceivably impact virulence regulation.
authors.affiliation: Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg, Denmark.
authors.forename: Kristoffer T
authors.initials: KT
authors.lastname: Bæk
authors.forename: Dorte
authors.initials: D
authors.lastname: Frees
authors.forename: Adriana
authors.initials: A
authors.lastname: Renzoni
authors.forename: Christine
authors.initials: C
authors.lastname: Barras
authors.forename: Natalia
authors.initials: N
authors.lastname: Rodriguez
authors.forename: Caroline
authors.initials: C
authors.lastname: Manzano
authors.forename: William L
authors.initials: WL
authors.lastname: Kelley
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D019295:Computational Biology; D014644:Genetic Variation / Q000235:genetics*; D016680:Genome, Bacterial / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D054643:INDEL Mutation / Q000235:genetics; D008969:Molecular Sequence Data; D010375:Pedigree; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013211:Staphylococcus aureus / Q000235:genetics*